PROJECT SUMMARY Patients with mesothelioma urgently need improved strategies to monitor treatment responses with systemic therapy and to detect early recurrence of disease after surgery. The growth patterns of mesothelioma and the therapeutic changes associated with its treatment limit the utility of imaging for these patients. Mesothelioma is a spatially complex disease that thickens along the pleura in circumferential sheets around the lungs. Standard imaging criteria used to assess treatment response were not designed with this malignancy in mind. Tumor measurements cannot be accurately made adjacent to non-malignant tissue such as the diaphragm or atelectasis or next to inflammation or scarring that result from common treatments such as surgery or talc pleurodesis. Whereas mesothelioma has very few nonsynonymous point mutations, we have discovered that large chromosomal rearrangements detected by Mate-Pair sequencing (MPseq) are common in this disease. Conventional sequencing approaches do not readily detect these rearrangements. In our preliminary data we show how these rearrangements can be detected in blood as cell-free tumor DNA (ctDNA). The objectives of our proposal are to optimize the selection of chromosomal rearrangements for detection in blood as ctDNA, and to determine whether longitudinal changes in cell-free chromosomal rearrangements correlate with imaging-based standards of response assessment for patients with mesothelioma. Our central hypothesis is that chromosomal rearrangements can be detected as ctDNA and that their changes will correlate with conventional radiographic criteria of response. We will test our working hypothesis by using the approach of prospective, correlative biomarker studies with the following two specific aims: (1) determine the extent to which circulating tumor chromosomal rearrangements can be detected in patients with newly diagnosed mesothelioma and (2) determine the extent to which the dynamics of chromosomal rearrangement ctDNA correlate with response to systemic chemotherapy as defined by conventional radiologic criteria. This approach is highly innovative because the low number of single nucleotide variants in mesothelioma precludes the use of commercial ctDNA assays for patients with this disease. Our preliminary data suggest that with optimization, the use of cell-free chromosomal rearrangements will overcome the limited sensitivities of other cell-free DNA tests for mesothelioma and provide a blood-based biomarker of therapeutic efficacy. This proposed research is significant because the successful completion of our study would improve upon our standards of care for disease monitoring and response assessment. Additionally, the use of chromosomal rearrangement ctDNA as a real-time biomarker of therapeutic efficacy could be incorporated into clinical trials to help physicians select the patients at highest risk of recurrence to receive systemic therapy while sparing toxicities in those who are at lower risk, and to accelerate drug development with early signals of efficacy.